Topology of Xer recombination on catenanes produced by lambda integrase.

Xer site-specific recombination at the psi site from plasmid pSC101 displays topological selectivity, such that recombination normally occurs only between directly repeated sites on the same circular DNA molecule. This intramolecular selectivity is important for the biological role of psi, and is imposed by accessory proteins PepA and ArcA acting at accessory DNA sequences adjacent to the core recombination site. Here we show that the selectivity for intramolecular recombination at psi can be bypassed in multiply interlinked catenanes. Xer site-specific recombination occurred relatively efficiently between antiparallel psi sites located on separate rings of right-handed torus catenanes containing six or more nodes. This recombination introduced one additional node into the catenanes. Antiparallel sites on four-noded right-handed catenanes, the normal product of Xer recombination at psi, were not recombined efficiently. Furthermore, parallel psi sites on right-handed torus catenanes were not substrates for Xer recombination. These findings support a model in which psi sites are plectonemically interwrapped, trapping a precise number of supercoils that are converted to four catenation nodes by Xer strand exchange.     

Genetic variation in the Bribri and Cabecar Amerindians from Talamanca, Costa Rica.

A screening of more than 40 loci was performed in the Bribri and Cabecar Amerindian populations of Talamanca in Southeastern Costa Rica. Some differences were found in the distribution (presence or absence) of the variants (some of them private) of the GOT, PEPA, RH, TF and TPI loci when the results reported here were compared with those of the Bribris and Cabecars that inhabit in the Pacific side of the country. Admixture with non-Indians was detected at the ABO, ADA, and G6PD loci. The proportion of polymorphic loci (P) found were 17.1 in the Bribris and 19.4 in the Cabecars; their average heterozygosites (H) estimates were 0.039 and 0.049 respectively. Fst values (0.019 in the Bribris and 0.056 in the Cabecars) seem to indicate that random events have played important roles in the divergence of the Cabecars. Low effective population sizes of the emigrant groups to the Pacific side and probable gene flow in the case of the Bribris, could be the cause of these differences.     

Small bowel and colon glucose absorption study in rats by an adaptation of Sols and Ponz method.

An adaptation of the Sols and Ponz method for the study of glucose intestinal absorption was developed by considering the special conditions of our line research. The glucose absorption was studied in proximal jejunum, distal ileum and distal colon in Wistar rat. The main adaptations in the method for successive absorptions with intestinal perfusions in vivo were the length of the intestinal segment and the change of the pumping system. The results are very similar to those obtained with the original method.     

L-thyroxine and L-triiodothyronine in the cerebral tissues of the adult rat. Effect of hypothyroidism]

The L-thyroxine and L-triiodothyronine concentrations in several brain areas (cerebral cortex, brain stem, hypothalamus, total brain and hypophysis) in normal and hypothyroid rats have been studied. Results show that L-thyroxine values at tissue level are inferior in the hypothyroid group, although non-significant with respect to the control group, whereas L-triiodothyronine presents values similar to the hypothyroid group and its control in all the brain regions studied with the exception of hypophysis. These results show that in hypothyroid situations exist a compensatory mechanism for maintaining the adequate L-triiodothyronine levels in several brain areas, although the serum levels are strongly decreased in hypothyroid animals.     

Thromboxane and prostacyclin generation by human umbilical veins after thrombin.

Prostacyclin (PGI2) and Thromboxane B2 (TxB2) production induced by thrombin in human umbilical veins (HUV) was studied. Successive stimulations of HUV segments were performed with and without restoration of arachidonic acid (AA). Thrombin consistently stimulated the production of both substances. The magnitude of the increment declined with progressive stimuli. The addition of exogenous AA could restore the production of TXB2 but not that of PGI2. These results suggest that sustained stimulation of AA release may lead to an imbalance in the TXA2/PGI2 ratio perhaps through an effect of unknown products of AA oxidation on PGI2 synthase.     

Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding.

Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities; native IGF-1 [18-61,6-48,47-52] and IGF-1 swap [18-61,6-47, 48-52]. Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae. IGF-1 swap had a approximately 30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1.The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-1 swap for the insulin receptor was approximately 200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor. A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region alpha-helix and a rearrangement in the relative orientation of the A- and B-regions. The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented. However, in the model of IGF-1 swap the C-region has filled the space where the first A-region alpha-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket.     

The influence of alkyl pyridinium sponge toxins on membrane properties, cytotoxicity, transfection and protein expression in mammalian cells

The ability of two alkyl pyridinium sponge toxin preparations (poly-APS and halitoxin) to form transient pores/lesions in cell membranes and allow transfection of plasmid cDNA have been investigated using HEK 293 cells. Poly-APS and halitoxin preparations caused a collapse in membrane potential, reductions in input resistance and increased Ca²⁺ permeability. At least partial recovery was observed after poly-APS application but recovery was more rarely seen with halitoxin. The transfection with plasmid cDNAs for an enhanced green fluorescent protein (EGFP) and human tumour necrosis factor receptor 2 (TNFR2) was assessed for both toxin preparations and compared with lipofectamine. Stable transfection was achieved with poly-APS although it was less efficient than lipofectamine. These results show that viable cells transfected with alien cDNA can be obtained using novel transient pore-forming alkyl pyridinium sponge toxins and a simple pre-incubation protocol. This provides the first proof of principle that pore-forming alkyl pyridinium compounds can be used to deliver cDNA to the intracellular environment without permanently compromising the plasma membrane.